The side-chain cleavage of cholesterol by CYP11A1 is not implicated in the biosynthesis of MBG, as it is for endogenous ouabain. Previous studies in toads, known to produce bufadienolides, demonstrated that administration of labeled cholesterol and cholanic acid, but not of the product of cholesterol side-chain cleavage, progesterone, resulted in the incorporation of a radioactive label into bufadienolide molecules. Because the extra-hepatic synthesis of bile acids has recently been described, we hypothesized that in placenta, in which high levels of bufadienolides are detected in preeclampsia, MBG could also be synthesized from bile acids. Thus, we examined the impact of silencing of two genes encoding, CYP27A1, a rate-limiting enzyme for synthesis of cholanic acid from cholesterol, and of CYP11A1, an enzyme which controls side-cleavage of cholesterol into pregnenolone, in human trophoblast JEG-3 cells on production of MBG and progesterone. Silencing of CYP11A1 markedly reduced production of progesterone but did not affect production of MBG by JAG3 cells. Silencing of CYP27A1, conversely, did not affect production of progesterone, but suppressed production of MBG. Thus, human trophoblast cells synthesize MBG from cholesterol via bile acid pathway.